Abstract Tumor-infiltrating T cell metabolic dysfunction and genetic reprogramming for effective immunotherapy The ability to target and destroy mutated cells is an essential characteristic of the immune system. Despite this attribute, the immune system largely fails to eliminate cancer once a tumor is established. This is due in part to the immunosuppressive nature of the tumor microenvironment (TME), which inhibits CD8+ tumor infiltrating T lymphocytes (CD8+ TIL) from becoming activated and killing their target cells. While it is known that the TME can directly inhibit CD8+ TIL through receptor-ligand interactions and suppressive cytokines to cause phenotypically and functionally exhausted T cells, it is increasingly understood that the TME is also metabolically suppressive. Tumor cells utilize an abundance of metabolites due to their proliferative nature, creating an additional suppressive mechanism for CD8+ TIL, as they need adequate nutrients for effector functions and to fuel their own proliferative nature. In this NCI Predoctoral to Postdoctoral Fellow Transition Award (F99/K00) application, the metabolic deficiencies of CD8+ TIL are described, showing CD8+ TIL experience significant loss of mitochondrial mass and function. This is due to repression of mitochondrial biogenesis transcription factor PGC1?, due to chronic Akt activation in TIL. When PGC1? was overexpressed in CD8+ TIL, it not only led to increased mitochondria, but improved TIL effector function and decreased tumor burden. These results led to the proposed studies: to further understand why mitochondria are important to TIL function. It is hypothesized that mitochondria are the defining organelle between a functional memory T cell and a dysfunctional exhausted T cell. To explore this hypothesis, T cells will be rendered metabolically- deficient in vitro through pharmacologic depletion of mitochondrial function. These metabolically deficient T cells (called Rho0 T cells) will be used to explore how mitochondria may be essential for calcium buffering to limit NFAT and an exhaustion genetic profile, as well as how mitochondria may be required for preventing an exhaustion epigenetic profile. After the completion of these studies, the applicant will transition to postdoctoral studies, where they will further explore TIL dysfunction, but with the goal of a translational, therapeutic outcome. The applicant will study how chimeric antigen receptor (CAR) T cells may be improved upon and used therapeutically in human T cells. Understanding how T cells become dysfunctional in cancer is important for both our understanding of T cell biology, and for improving targeted cancer immunotherapy.